This invention relates to novel immunoreactive phospholipid conjugates useful for homogeneous lipsome immunoassays. More particularly, this invention provides a rapid and efficient approach to digoxin and digoxin-related phospholipid conjugates which are useful for liposome immunoassay. In one novel aspect, a synthetic procedure is provided which involves the linking of a terminal sugar group, e.g., digitoxose of digoxin, to a phospholipid through a carboxymethyl oxime functionality. Such procedure provides much improved yields of readily purified products compared to known procedures. Moreover, it is applicable to related phospholipid conjugates where linkage through a sugar ring is highly preferred, i.e. digitoxin, gitoxin, ouabain, digitonin and other related cardiac glycosides. In addition to these cardiac glycosides, other glycosides including those from the saponin class, but not limited to that class, are within the purview of this invention.
Still further, the method of this invention is also applicable to phospholipid conjugates derived from steroids such as estrogens and testosterones which can be modified to form oxime derivatives remote from key functionalities important for immunorecognition by specific antibodies. The novel phospholipid conjugates disclosed herein are useful for sensitive liposome immunoassays, e.g., for digoxin.
Digoxin is a potent cardiac glycoside. Toxic amounts of digoxin exert undesirable and potentially lethal electrophysiological effects [Hoffman et al, The Pharmacological Basis of Therapeutics, Gilman, 6th ed., p 729, New York 1980]. Accordingly, various immunoassay methods for cardiac glycosides are now widely used clinically as aids in the determination of appropriate dosage schedules for patients receiving these drugs. Because digoxin is too small a molecule to be antigenic by itself, it is necessary to conjugate digoxin convalently as a hapten to antigenic carriers, for example, human serum albumin (HSA), bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) in order to elicit digoxin-specific antibodies in experimental animals for use in immunoassay. The preparation of immunoreactive digoxin derivatives is typically carried out by the procedure of Butler et al [Proc. Natl. Acad. Sci., U.S.A., 1967, 57, 71-78; and Methods in Enzymology, Academic Press, 84, 558-577 (1982)] which is based on the work of Erlanger and Beiser, Proc. Natl. Acad. Sci., 52, 68 (1964). The reaction sequence involved periodate cleavage of the terminal sugar ring (digitoxose or rhamnose) followed by reaction with a protein carrier, enzyme or related biological molecule, and finally reductive amination with sodium borohydride. Thus, digoxin-HSA (Butler et al supra), digoxin-BSA [Smith et al, Biochemistry, 9, 331-337 (1970)], mellitin-Ouabain [Freytag et al, J. of Immunological Methods, 70, 133-140 (1984)], and digoxin-dibenzo-18-crown-6 [Keating et al, Anal. Chem. 56, 801-806 (1984)] conjugates have been prepared using the aforementioned reaction sequence.
U.S. Pat. No. 4,115,539 discloses a method of preparing digoxin conjugates using isocyanates based upon tyrosine methyl ester. U.S. Pat. Nos. 4,297,273 and 4,363,759 disclose ways to prepare chemiluminescent phthalaldehyde-labeled digoxigenin. U.S. Pat. No. 4,342,826 employs the procedure of Butler et al supra to prepare a non-characterized digoxin-phosphatidylethanolamine conjugate but, this preparation in our hands, resulted in a very complex mixture.
Hence, the present invention satisfies a strong need for digoxin and digoxin-related phospholipid conjugates and other conjugates useful for sensitive liposome immunoassays.
The novel synthesis, disclosed herein, involves the linking of a dialdehyde intermediate, e.g. digoxin dialdehyde, through a carboxymethyl oxime functionality resulting in significantly high yields of readily purified products.